Volatile Metabolite Profiles Research Articles

Dynamic trajectories of volatile and non‐volatile specialised metabolites in ‘overnight’ fragrant flowers of Murraya paniculata

Ephemeral flowers, especially nocturnal ones, usually emit characteristic scent profiles within their post-anthesis lifespans of a few hours. Whether these flowers exhibit temporal variability in the composition and profile of volatile and non-volatile specialised metabolites has received little attention. Flowers of Murraya paniculata bloom in the evenings during the summer and monsoon, and their sweet, intense fragrance enhances the plant's value as an ornamental. We aimed to investigate profiles of both volatile and non-volatile endogenous specialised metabolites (ESM) in nocturnal ephemeral flowers of M.paniculata to examine whether any biochemically diverse groups of ESM follow distinct patterns of accumulation while maintaining synchrony with defensive physiological functions. Targeted ESM contents of M.paniculata flowers were profiled at ten time points at 2-h intervals, starting from late bud stage (afternoon) up to the start of petal senescence (mid-morning). Emitted volatiles were monitored continuously within the whole 20-h period using headspace sampling. The ESM contents were mapped by time point to obtain a highly dynamic and biochemically diverse profile. Relative temporal patterns of ESM accumulation indicated that the active fragrance-emitting period might be divided into 'early bloom', 'mid-bloom' and 'late bloom' phases. Early and late bloom phases were characterised by high free radical generation, with immediate enhancement of antioxidant enzymes and phenolic compounds. The mid-bloom phase was relatively stable and dedicated to maximum fragrance emission, with provision for strong terpenoid-mediated defence against herbivores. The late bloom phase merged into senescence with the start of daylight; however, even the senescent petals continued to emit fragrance to attract diurnal pollinators. Our study suggests that dynamic relations between the different ESM groups regulate the short-term requirements of floral advertisement and phytochemical defence in this ephemeral flower. This study also provided fundamental information on the temporal occurrence of emitted volatiles and internal pools of specialised metabolites in M.paniculata flowers, which could serve as an important model for pollination biology of Rutaceae, which includes many important fruit crops.

Profiling of volatile and non-volatile metabolites in Polianthes tuberosa L. flowers reveals intraspecific variation among cultivars

Polianthes tuberosa L. (tuberose) is a widely cultivated ornamental crop in Asian countries. Different cultivars of tuberose have been developed through breeding programs in India. However, no reports on floral fragrance and metabolite contents of these cultivars are available. In this study, an attempt has been made to evaluate the levels of both volatile and non-volatile metabolites from seven different cultivars of P. tuberosa. Presence of benzenoids, phenylpropanoids, terpenoids, and few fatty acid derivatives as emitted, endogenous and glycosylated forms were revealed from the studied cultivars. Further, chemometric analyses in both supervised and unsupervised manner led to identification of patterns among the cultivars. Among the seven cultivars, four distinct clusters were obtained linking to their volatiles, flavonoids and primary metabolite levels. Metabolic variations obtained from the cultivars also suggest cross-talks between phenylpropanoid, benzenoid, and flavonoid pathways. Thus metabolite profiling reported here may help in characterization of tuberose cultivars for perfumery utility and future breeding programme.

Rapid discrimination of fungal strains isolated from human skin based on microbial volatile organic profiles

Studies of the microbiota of human skin have gained attention mainly because of its high complexity. Volatile metabolites that emerge from the microbiota play a significant role in fungus metabolism, acting on fungal development, defense, and protection against stress, communication, and pathogenicity. The present study evaluated volatile organic profiles, based on headspace–solid-phase microextraction–gas chromatography–mass spectrometry. We sought to define the optimal experimental conditions for such identification. Chromatograms from 15 fungi were evaluated and discriminated by principal component analysis. The volatile metabolite profiles that were putatively identified in the present study (e.g 2‑isopropyl‑5‑methyl‑cyclohex‑3‑en‑1‑one, 3/2‑methyl‑1‑butanol, isopentyl ethanoate, phenyl ethanol) allowed the discrimination of different microorganisms from human skin. The present methodology may be a more rapid way of identifying microorganisms compared with conventional methods of microbiological identification.

Sniffing out resistance – Rapid identification of urinary tract infection-causing bacteria and their antibiotic susceptibility using volatile metabolite profiles

Antibiotic resistance is set to be an unprecedented threat to modern medicine. ‘Sniffing’ bacteria potentially offers a rapid way to determine susceptibility. A successful proof-of-principle study is described, using thermal desorption-gas chromatography-mass spectrometry (TDGCMS) to ‘smell’ cephalexin and ciprofloxacin resistant and sensitive Urinary Tract Infection (UTI)-causing bacteria. 578 peaks at unique retention times were detected from 86 chromatograms of 18 bacterial isolates (E. coli, K. pneumoniae and P. aeruginosa). The isolates were grown with and without the presence of antibiotic. Chi-square analysis found 9 compounds that differed significantly between cephalexin sensitive and resistant isolates, and 22 compounds that differed significantly between ciprofloxacin sensitive and resistant isolates, at p ≤ 0.05. When antibiotic was added to the media, more differences were found in the cephalexin group, attributed to lysis, but not in the ciprofloxacin group. Further work with large sample sizes will potentially enable the development of diagnostic algorithms using presence/absence of particular compounds of interest.

Multiple stir bar sorptive extraction combined with gas chromatography-mass spectrometry analysis for a tentative identification of bacterial volatile and/or semi-volatile metabolites

We propose a new approach combining the principles and advantages of stir bar sorptive extraction (SBSE) and headspace sorptive extraction (HSSE). Stir bars have so far never been used for the extraction of volatile/semi-volatile bacterial compounds. The effectiveness of two stir bars with polydimethylsiloxane (PDMS) or ethylene glycol/silicone (EGS) as sorbent was tested by performing sample extraction directly in gas chromatography (GC) vials containing bacterial cultures. Several combinations of desorption and extraction were tested at different growth times. When the extraction was carried out simultaneously with the EGS stir bar in headspace and the PDMS in the bacterial culture, the number of extracted compounds was significantly increased. Using both twisters increased the polarity range of the compounds found, and extraction at the end of the exponential phase of growth generated the best yields. This method was successfully applied to determine the production of 17 molecules by a strain of Staphylococcus aureus. In conclusion, this study paves the way for a new method for determining the volatile metabolite profile of bacteria, which can provide a promising innovative alternative in the identification of biomarkers.

Volatile metabolite profiling of ten Hedychium species by gas chromatography mass spectrometry coupled to chemometrics

The Hedychium genus (Zingiberaceae) produces a plethora of secondary metabolites that include terpenoids, some of which are known for their aromatic properties. The volatile compositions of several members of this genus have not been chemically defined, as many species are endemic to remote ecosystems. In the present research, the essential oil composition of ten Hedychium species (H. gardnerianum, H. flavescens, H. thyrisiforme, H. flavum, H. ellipticum, H. aurantiacum, H. gracile, H. greenii, H. spicatum, and H. coronarium) was studied by gas chromatography flame ionization detector (GC-FID) and gas chromatography mass spectrometry (GCMS). The essential oil yield obtained by hydrodistillation varied from 0.05 (%v/w) in H. greenii to 0.75 (%v/w) in H. ellipticum and H. thyrsiforme, respectively. A total of 56 constituents representing 75.93–99.66% of volatile constituents were characterized in the essential oil of 10 Hedychium species. The predominant components were β-pinene in H. gardnerianum, H. gracile, H. thyrsiforme, H. flavum and H. flavescens; bornyl acetate in H. greenii; humulene epoxide I in H. aurantiacum; 1,8-cineole in H. spicatum and H. coronarium. Agglomerative Hierarchical Clustering (AHC) and Principal component analysis (PCA) based on essential oil compositions grouped Hedychium species into three different clusters. In view of the ambiguity existing among different Hedychium species, the developed chemical fingerprint in the present study would assume great significance in identification and classification of Hedychium species.

GC-MS metabolomics reveals disturbed metabolic pathways in primary mouse hepatocytes exposed to subtoxic levels of 3,4-methylenedioxymethamphetamine (MDMA).

3,4-Methylenedioxymethamphetamine (MDMA, ecstasy) is a well-known hepatotoxic drug. Although its toxicity has been thoroughly studied at high concentrations, there is still insufficient knowledge on possible alterations of cell function at subtoxic concentrations, which are in fact more representative concentrations of intoxication scenarios. In this study, a gas chromatography-mass spectrometry (GC-MS) metabolomics approach was used to investigate the metabolic changes in primary mouse hepatocytes (PMH) exposed to two subtoxic concentrations of MDMA (LC01 and LC10) for 24h. Metabolomic profiling of both intracellular metabolites and volatile metabolites in the extracellular medium of PMH was performed. Multivariate analysis showed that the metabolic pattern of cells exposed to MDMA discriminates from the controls in a concentration-dependent manner. Exposure to LC10 MDMA induces a significant increase in some intracellular metabolites, including oleic acid and palmitic acid, and a decrease in glutamate, aspartate, 5-oxoproline, fumarate, malate, phosphoric acid, α-ketoglutarate and citrate. Extracellular metabolites such as acetophenone, formaldehyde, pivalic acid, glyoxal and 2-butanone were found significantly increased after exposure to MDMA, compared to controls, whereas 4-methylheptane, 2,4-dimethyl-1-heptene, nonanal, among others, were found significantly decreased. The panel of discriminatory metabolites is mainly involved in tricarboxylic acid (TCA) cycle, fatty acid metabolism, glutamate metabolism, antioxidant defenses and possibly changes in the liver enzyme machinery. Overall, these results highlight the potential of the intra- and extracellular metabolome to study alterations triggered by subtoxic concentrations of MDMA in hepatic cell functions, which represents a more realistic appraisal of early toxicity events posed by exposure to this drug. In addition, these results also revealed some metabolites that may be used as potential biomarkers indicative of early events in the hepatotoxicity induced by MDMA.

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (Formicidae) for Volatilome Analysis by GC-MS and MetaboliteDetector.

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean 'exploding ants' belonging to the Colobopsis cylindrica (COCY) group. For this purpose, the model species C. explodens is used. Ants belonging to the minor worker caste possess distinctive hypertrophied mandibular glands (MGs). In territorial combat, they use the viscous contents of their enlarged mandibular gland reservoirs (MGRs) to kill rival arthropods in characteristic suicidal 'explosions' by voluntary rupture of the gastral integument (autothysis). We show the dissection of worker ants of this species for the isolation of the gastral portion of the wax-like MGR contents as well as listing the necessary steps required for solvent-extraction of the therein contained volatile compounds with subsequent gas chromatography-mass spectrometry (GC-MS) analysis and putative identification of metabolites contained in the extract. The dissection procedure is performed under cooled conditions and without the use of any dissection buffer solution to minimize the changes in the chemical composition of the MGR contents. After solvent-based extraction of volatile metabolites contained therein, the necessary steps for analyzing the samples via liquid-injection-GC-MS are presented. Lastly, data processing and putative metabolite identification with the use of the open-source software MetaboliteDetector is shown. With this approach, the profiling and identification of volatile metabolites in MGRs of ants belonging to the COCY group via GC-MS and the MetaboliteDetector software become possible.

Isolation of Mandibular Gland Reservoir Contents from Bornean 'Exploding Ants' (<em>Formicidae</em>) for Volatilome Analysis by GC-MS and MetaboliteDetector

The aim of this manuscript is to present a protocol describing the metabolomic analysis of Bornean 'exploding ants' belonging to the Colobopsis cylindrica (COCY) group. For this purpose, the model species C. explodens is used. Ants belonging to the minor worker caste possess distinctive hypertrophied mandibular glands (MGs). In territorial combat, they use the viscous contents of their enlarged mandibular gland reservoirs (MGRs) to kill rival arthropods in characteristic suicidal 'explosions' by voluntary rupture of the gastral integument (autothysis). We show the dissection of worker ants of this species for the isolation of the gastral portion of the wax-like MGR contents as well as listing the necessary steps required for solvent-extraction of the therein contained volatile compounds with subsequent gas chromatography-mass spectrometry (GC-MS) analysis and putative identification of metabolites contained in the extract. The dissection procedure is performed under cooled conditions and without the use of any dissection buffer solution to minimize the changes in the chemical composition of the MGR contents. After solvent-based extraction of volatile metabolites contained therein, the necessary steps for analyzing the samples via liquid-injection-GC-MS are presented. Lastly, data processing and putative metabolite identification with the use of the open-source software MetaboliteDetector is shown. With this approach, the profiling and identification of volatile metabolites in MGRs of ants belonging to the COCY group via GC-MS and the MetaboliteDetector software become possible.

In vivo investigation of the volatile metabolome of antiphytopathogenic yeast strains active against Penicillium digitatum using comprehensive two-dimensional gas chromatography and multivariate data analysis

Penicillium digitatum is the responsible for green mold decay on citrus fruits, which contributes to economic losses in agriculture industry during storage, transportation and in the market. Biocontrol agents may be a viable and low-cost alternative in the control of different plant pathogens: metabolites from some yeast strains can prevent infection, decreasing host tissue colonization and reducing pathogen survival and sporulation with varying degrees of efficiency. Eight S. cerevisae strains and one Candida stellimalicola strain all were evaluated in vitro as biological control agents against P. digitatum. After in vitro tests the headspace of yeast strains were extracted in vivo using an optimized HS-SPME + GC × GC–MS method. The correlation between antiphytopathogenic activity with the volatile metabolite profiles was assessed using principal component analysis. Nine compounds were associated with the volatile metabolome of strains with higher antifungal action: ethyl propionate, 3-methyl-1-butanol, 2-methyl-1-butanol, ethyl butyrate, 3-methylbutyl acetate, 3-methylbutyl propionate, n-pentyl butyrate, phenylethyl alcohol and 2-phenethyl acetate. This study allowed for the first time a comprehensive overview of the effect of individual volatile metabolites from yeast strains on P. digitatum infection.

In vivo investigation of the volatile metabolome of antiphytopathogenic yeast strains active against Penicillium digitatum using comprehensive two-dimensional gas chromatography and multivariate data analysis

Penicillium digitatum is the responsible for green mold decay on citrus fruits, which contributes to economic losses in agriculture industry during storage, transportation and in the market. Biocontrol agents may be a viable and low-cost alternative in the control of different plant pathogens: metabolites from some yeast strains can prevent infection, decreasing host tissue colonization and reducing pathogen survival and sporulation with varying degrees of efficiency. Eight S. cerevisae strains and one Candida stellimalicola strain all were evaluated in vitro as biological control agents against P. digitatum. After in vitro tests the headspace of yeast strains were extracted in vivo using an optimized HS-SPME + GC × GC–MS method. The correlation between antiphytopathogenic activity with the volatile metabolite profiles was assessed using principal component analysis. Nine compounds were associated with the volatile metabolome of strains with higher antifungal action: ethyl propionate, 3-methyl-1-butanol, 2-methyl-1-butanol, ethyl butyrate, 3-methylbutyl acetate, 3-methylbutyl propionate, n-pentyl butyrate, phenylethyl alcohol and 2-phenethyl acetate. This study allowed for the first time a comprehensive overview of the effect of individual volatile metabolites from yeast strains on P. digitatum infection.

Comparison of volatile and non-volatile metabolites in rice wine fermented by Koji inoculated with Saccharomycopsis fibuligera and Aspergillus oryzae

This study investigated volatile and nonvolatile metabolite profiles of makgeolli (a traditional rice wine in Korea) fermented by koji inoculated with Saccharomycopsis fibuligera and/or Aspergillus oryzae. The enzyme activities in koji were also examined to determine their effects on the formation of metabolites. The contents of all 18 amino acids detected were the highest in makgeolli fermented by S. fibuligera CN2601-09, and increased after combining with A. oryzae CN1102-08, unlike the contents of most fatty acids. On the other hand, major volatile metabolites were fusel alcohols, acetate esters, and ethyl esters. The contents of most fusel alcohols and acetate esters were the highest in makgeolli fermented by S. fibuligera CN2601-09, for which the protease activity was the highest, leading to the largest amounts of amino acods. The makgeolli samples fermented only by koji inoculated with S. fibuligera could be discriminated on PCA plots from the makgeolli samples fermented in combination with A. oryzae. In the case of nonvolatile metabolites, all amino acids and some metabolites such as xylose, 2-methylbenzoic acid, and oxalic acid contributed mainly to the characteristics of makgeolli fermented by koji inoculated with S. fibuligera and A. oryzae. These results showed that the formations of volatile and nonvolatile metabolites in makgeolli can be significantly affected by microbial strains with different enzyme activities in koji. To our knowledge, this study is the first report on the effects of S. fibuligera strains on the formation of volatile and non-volatile metabolites in rice wine, facilitating their use in brewing rice wine.

Nuclear magnetic resonance- and gas chromatography/mass spectrometry-based metabolomic characterization of water-soluble and volatile compound profiles in cabbage vinegar

Non-targeted metabolomic analyses employing nuclear magnetic resonance- and gas chromatography/mass spectrometry-based techniques were applied for an in-depth characterization of cabbage vinegar, an original agricultural product made from cabbage harvested in Tsumagoi, Japan. Water-soluble and volatile metabolite profiles of cabbage vinegar were compared with those of various vinegars: rice vinegar, grain vinegar, apple vinegar, and black vinegar (Japanese kurozu made of brown rice). Principal component analysis (PCA) of the water-soluble metabolites indicated that cabbage vinegars belonged to an isolated class by the contributions of fructose, pyroglutamic acid, choline, and methiin (S-methylcysteine sulfoxide). Regarding the volatile compounds, the PCA data represented that rice, black, and apple vinegars were characterized by most of the dominant volatiles, such as acetate esters, alcohols, ketones, and acids. Cabbage and grain vinegars were included in the same class although these two vinegars have different flavors. Orthogonal partial least squares-discrimination analysis exhibited the differences in volatile compound profile between cabbage and grain vinegars, revealing that cabbage vinegars were characterized by the presence of sulfides (dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide), nitriles (allyl cyanide and 4-methylthio-butanenitrile), 3-hexene-1-ol, and crotonic acid. The time-course changes in these highlighted compounds during the acetic acid fermentation of cabbage vinegar suggested that pyroglutamic and crotonic acids were produced through fermentation, whereas choline, methiin, sulfides, nitriles, and 3-hexene-1-ol were derived from cabbage, suggesting the key role of these compounds in the unique taste and flavor of cabbage vinegar.

Modification of chrysanthemum odour and taste with chrysanthemol synthase induces strong dual resistance against cotton aphids.

SummaryAphids are pests of chrysanthemum that employ plant volatiles to select host plants and ingest cell contents to probe host quality before engaging in prolonged feeding and reproduction. Changes in volatile and nonvolatile metabolite profiles can disrupt aphid–plant interactions and provide new methods of pest control. Chrysanthemol synthase (CHS) from Tanacetum cinerariifolium represents the first committed step in the biosynthesis of pyrethrin ester insecticides, but no biological role for the chrysanthemol product alone has yet been documented. In this study, the TcCHS gene was over‐expressed in Chrysanthemum morifolium and resulted in both the emission of volatile chrysanthemol (ca. 47 pmol/h/gFW) and accumulation of a chrysanthemol glycoside derivative, identified by NMR as chrysanthemyl‐6‐O‐malonyl‐β‐D‐glucopyranoside (ca. 1.1 mM), with no detrimental phenotypic effects. Dual‐choice assays separately assaying these compounds in pure form and as part of the headspace and extract demonstrated independent bioactivity of both components against the cotton aphid (Aphis gossypii). Performance assays showed that the TcCHS plants significantly reduced aphid reproduction, consistent with disturbance of aphid probing activities on these plants as revealed by electropenetrogram (EPG) studies. In open‐field trials, aphid population development was very strongly impaired demonstrating the robustness and high impact of the trait. The results suggest that expression of the TcCHS gene induces a dual defence system, with both repellence by chrysanthemol odour and deterrence by its nonvolatile glycoside, introducing a promising new option for engineering aphid control into plants.

Volatile Metabolomic Composition of Vitex Species: Chemodiversity Insights and Acaricidal Activity.

The Vitex genus (Lamiaceae) produces a plethora of metabolites that include ecdysteroids and terpenoids, some of which have demonstrated insect repellent properties. The volatile composition of several members of this genus has not been chemically defined, as many taxa are endemic to remote ecosystems. In this study, leaves were collected from the northeast of Brazil from Vitex capitata, V. megapotamica, V. gardneriana, and V. rufescens plants and examined for their chemical profile via GC-MS/FID of essential oil extracts. The analyses showed a diversity of terpenoids. Of particular note were seven-member ring sesquiterpenes which were present in great abundance; a dendrogram showed clades separating by the production of bicyclogermacrene, aromadendrane and 5,10-cycloaromadendrane sesquiterpenoids for the four species. Comparison of volatile metabolite profiles to 13 other Vitex species showed strong similarities in the production of some monoterpenes, but varied by their production of larger terpenes, especially those with gem-dimethylcyclopropyl subunits on seven-member ring compounds. From this work, we suggest that the sesquiterpene skeleton with seven member rings is a good chemosystematic biomarker candidate for the Vitex genus. Separation using this biomarker was then validated using Inter-Simple Sequence Repeat profiling. Lastly, experiments examining the toxicity of these four oils against the coconut mite Aceria guerreronis showed that only the oil of V. gardneriana had strong acaricidal activity, with an LC50 of 0.85 mg/mL, thus demonstrating its potential for use as a natural pesticide.

Breath-Based Diagnosis of Invasive Mucormycosis (IM)

BackgroundTimely diagnosis of IM remains a major challenge in clinical mycology. Because of the lack of specific diagnostic methods for IM and the frequently fulminant nature of this infection, IM-associated mortality remains high.MethodsWe examined breath volatile metabolite profiles in a neutropenic murine model of IM, using the 3 Mucorales species that most commonly cause human IM - Rhizopus arrhizus var. arrhizus, R. arrhizus var. delemar, and R. microsporus - and for comparison, Aspergillus fumigatus. We infected female balb/c mice (N = 4 per group) treated with cyclophosphamide and cortisone followed by intranasal administration of 106 conidia of each species. 3 days post-infection, we collected breath samples from each mouse via tracheostomy using a flexiVent murine ventilator, examining breath volatile metabolites using thermal desorption gas chromatography/tandem mass spectrometry (GC-MS/MS). We also sampled breath prospectively from five patients eventually diagnosed with proven IM caused by R. microsporus, analyzing breath volatile metabolites using thermal desorption GC-MS/MS.ResultsEach Mucorales species produced a consistent profile of breath sesquiterpene secondary metabolite VOCs in our murine models, which distinguished these species from each other and from murine invasive aspergillosis (Figure A). These fungi shifted their secondary metabolism significantly in vivo, compared with their previously characterized in vitro metabolism. We found overlapping VOC sesquiterpene metabolites between breath samples from the murine model of R. microsporus infection and 5 of 5 patients with R. microsporus IM, with additional sesquiterpene secondary metabolites detected in patient breath, compared with the murine IM model (Figure B). In one patient with serial breath samples, these sesquiterpenes declined in abundance and disappeared with antifungal therapy, in parallel with clinical improvement (Figure C).ConclusionThe three Mucorales species that cause most human IM have distinct breath sesquiterpene profiles that can be used to identify these infections in vivo noninvasively. These profiles distinguish these infections from each other and from aspergillosis, and may be useful in monitoring clinical response to treatment.Disclosures F. M. Marty, Astellas Pharma US: Consultant and Grant Investigator, Consulting fee and Grant recipient; Chimerix: Consultant and Grant Investigator, Consulting fee and Grant recipient; Fate Therapeutics: Scientific Advisor, Consulting fee; Gilead Sciences: Consultant and Grant Investigator, Consulting fee and Grant recipient; LFB: Consultant, Consulting fee; Merck: Consultant, Grant Investigator and Scientific Advisor, Consulting fee and Grant recipient; Roche Molecular Systems: Consultant, Consulting fee; Shire: Consultant and Grant Investigator, Consulting fee and Grant recipient; D. P. Kontoyiannis, Pfizer: Research Contractor, Research support and Speaker honorarium; Astellas: Research Contractor, Research support and Speaker honorarium; Merck: Honorarium, Speaker honorarium; Cidara: Honorarium, Speaker honorarium; Amplyx: Honorarium, Speaker honorarium; F2G: Honorarium, Speaker honorarium

Exhaled Volatile Organic Compounds of Infection: A Systematic Review.

With heightened global concern of microbial drug resistance, advanced methods for early and accurate diagnosis of infection are urgently needed. Analysis of exhaled breath volatile organic compounds (VOCs) toward detecting microbial infection potentially allows a highly informative and noninvasive alternative to current genomics and culture-based methods. We performed a systematic review of research literature reporting human and animal exhaled breath VOCs related to microbial infections. In this Review, we find that a wide range of breath sampling and analysis methods are used by researchers, which significantly affects interstudy method comparability. Studies either perform targeted analysis of known VOCs relating to an infection, or non-targeted analysis to obtain a global profile of volatile metabolites. In general, the field of breath analysis is still relatively immature, and there is much to be understood about the metabolic production of breath VOCs, particularly in a host where both commensal microflora as well as pathogenic microorganisms may be manifested in the airways. We anticipate that measures to standardize high throughput sampling and analysis, together with an increase in large scale collaborative international trials, will bring routine breath VOC analysis to improve diagnosis of infection closer to reality.

Alterations in volatile metabolites profile of fresh tomatoes in response to Alternaria alternata (Fr.) Keissl. 1912 infection

Alternaria alternata (Fr.) Keissl. 1912 is one of the main fungal pathogens that infect tomato (Solanum lycopersicum L.) during cold storage affecting postharvest quality and marketing. During fungal infections, fruits and fungi release specific volatile metabolites (VM) that could alter the fruit aroma, or could mediate resistance response in the fruit, or they also could suggest the possible status of fungal attack. The detection of the VM released during the tomato-Alternaria interaction could contribute to the development of ecofriendly and harmless strategies for its control. In this study, the profile of VM of fresh tomatoes inoculated with A. alternata, were analyzed by solid phase microextraction and gas chromatography-mass spectrometry (SPME-GC-MS) during storage at 15 and 20 °C for 48 h, respectively. Changes in the profile of VM were observed between control and inoculated fruit since the first few hour post-inoculation. Some VM (3-methyl-2-butenal, dimethyl disulfide, 1-butenol, hexanol, 2-methyl-1-butanol acetate, among others) were only detected in inoculated fruit, so they appear to be synthesized by the presence of the pathogen. Also, a marked increase of 3-methyl-1-butanol and 6-methyl-5-hepten-1-one were observed in inoculated fruit, and they were progressive over time particularly at 20 °C. In conclusion, A. alternata induced changes in the profile of volatile metabolites released by tomato fruit. Some of the VM released during tomato-A. alternata interaction, were synthesized or stimulated by the fungal attack. These results contribute to the current knowledge about the profile of VM released during the fruit-pathogen interaction.

Volatile metabolite profiling reveals the changes in the volatile compounds of new spontaneously generated loquat cultivars

In recent years, the advantageous traits of three new loquat cultivars have drawn the attention of breeders and growers. All three have spontaneously arisen from the ‘Algerie’ cultivar: the new ‘Xirlero’ cultivar is a bud mutant of ‘Algerie’, while ‘Amadeo’ and ‘Raúl’ arose as chance seedlings. Following a non-targeted approach based on HS-SPME-GC–MS, the volatile compounds profile of the fruits from the new cultivars were obtained and compared to the original ‘Algerie’ cultivar. Carboxylic acids clearly dominated the volatile profile of all the loquat cultivars, but esters, aldehydes, ketones and alcohols were also predominant compounds. Interestingly when the bud mutant event did not lead to marked changes in the volatile compounds complement, pronounced changes in the volatile composition of chance seedling-generated cultivars ‘Amadeo’ and ‘Raúl’ were observed. ‘Amadeo’ fruits showed lower levels of 2-methyl butanoic acid and much higher levels of methylhexanoate, methylbutanoate and 2-hydroxy-5-methylacetophenone. The ‘Raúl’ cultivar also had a distinctive volatile profile characterised by high levels of C6-aldehydes, (E)-2-hexanal, 2-hexenal, (Z)-3-hexenal and hexanal, and several carotenoid-derived volatiles; e.g. 2-pentene-1,4-dione 1-(1,2,2-trimethylcyclopentyl), (S)-dihydroactinidiolide, isodurene, cis-geranyl acetone, β-damascenone, β-ionone, α-ionone and 3,4-dehydro-β-ionone. These changes in volatiles were associated with a more intense flavour in cultivars ‘Amadeo’ and ‘Raúl’, according to the sensory evaluation of the flavour intensity carried out by a semi-trained panel. A metabolomic correlation network analysis provided insights as to how volatiles were regulated, and revealed that the compounds modified in ‘Amadeo’ were uncoupled from the rest of the volatilome, while the volatiles modified in ‘Raul’ changed according to specific groups. To conclude, this work provides a holistic view of how the loquat volatilome was affected, and this information was integrated with the physical-chemical-sensory attributes to understand the changes that occur in the new cultivars.

Photosynthetic performance and volatile organic compounds profile in Eucalyptus globulus after UVB radiation

Economically important forest species as Eucalyptus globulus face risks associated with climate changes, whose immediate and long-term damages are yet to account. The aim of this study was to decipher the impacts of UV-B radiation on the physiological performance and metabolome profile of leaves of Eucalyptus juvenile plants. For that, three-month-old plants were treated for two days to a total effective radiation of 12kJ m−2. One and 11 d after treatment the samples were collected for photosynthetic analyses. UV-B did not affect the photochemical efficiency or gas exchange, but the total soluble sugars (TSS), starch levels and pigment contents were changed. Changes in TSS and pigment contents persisted in time after the end of exposure. The profile of volatile metabolites (1 and 11 days after stress removal) was analyzed by HS-SPME/GC-IT-MS, and showed abundance of monoterpenes, sesquiterpenes and aldehydes. One day after the stress removal, an increase of aldehydes was observed and a decrease of some monoterpenes and sesquiterpenes. Eleven days after stress removal, some monoterpenes and sesquiterpenes decreased to values closer to those of control. The absence of mortality, and the minor effects observed in photosynthesis suggest a high tolerance profile of this species to the UV-B dose used, which may be justified by changes at the level of primary metabolism (involving sugars) and secondary (e.g. terpenes) observed in response to UV's. The correlations of these changes with potential functional adaptations are discussed here.